Receptor specific transepithelial transport of therapeutics

ABSTRACT

The present invention relates in general to methods and products for initiating an immune response against an antigen, and in particular relates to transepithelial delivery of antigens to provoke tolerance and immunity. The present invention further relates to methods and products for the transepithelial delivery of therapeutics. In particular, the invention relates to methods and compositions for the delivery of therapeutics conjugated to a FcRn binding partner to intestinal epithelium, mucosal epithelium and epithelium of the lung. The present invention further relates to the synthesis, preparation and use of the FcRn binding partner conjugates as, or in, pharmaceutical compositions for oral systemic delivery of drugs and vaccines.

This is a continuation of U.S. Ser. No. 10/215,298, filed Aug. 8, 2002(now U.S. Pat. No. 7,067,129), which is a division of U.S. Ser. No.09/122,144, filed Jul. 24, 1998 (now U.S. Pat. No. 6,485,726), which isa continuation-in-part of and claims priority to U.S. Ser. No.08/899,856, filed Jul. 24, 1997 (now U.S. Pat. No. 6,030,613), which isa continuation-in-part of U.S. Ser. No. 08/578,171, filed on Dec. 29,1995 (abandoned), which is a continuation-in-part of U.S. Ser. No.08/374,159, filed on Jan. 17, 1995 (now U.S. Pat. No. 6,086,875).

The work described herein was supported, in part, by National Institutesof Health Grant Nos. NIH DK-44319, NIH HO-27691 and NIH DK-48106. TheU.S. Government has certain rights to this invention.

1. FIELD OF THE INVENTION

The present invention relates in general to methods and products forinitiating an immune response against an antigen, and in particularrelates to transepithelial delivery of antigens to provoke tolerance andimmunity.

The present invention further relates to methods and products for thetransepithelial delivery of therapeutics. In particular, the inventionrelates to methods and compositions for the delivery of therapeuticsconjugated to a FcRn binding partner to intestinal epithelium, mucosalepithelium and epithelium of the lung. The present invention furtherrelates to the synthesis, preparation and use of the FcRn bindingpartner conjugates as, or in, pharmaceutical compositions for oralsystemic delivery of drugs and vaccines.

2. BACKGROUND OF THE INVENTION

The immune system of a mammal develops during gestation and becomesactive in the late mammalian fetus. Although active, it still might becharacterized as ‘immature’ because it has not been challenged to anysignificant extent by antigens; the fetus is largely protected fromantigens by the mother. This ‘immature’ immune system, however, issupplemented by the transfer of maternal immunoglobulin to the fetus (orin some cases to the neonate) to provide humoral immunity during thefirst weeks of independent life.

Rats and mice receive most maternal immunoglobulin G (IgG) by sucklingfrom colostrum and milk, although some is acquired prenatally. Cattlealso receive IgG from colostrum. In rabbits, IgG is transported to thefetus across the yolk sac. Little is know about the transfer of IgG tothe fetus or neonate in humans. Most evidence suggests that humanmothers transfer humoral immunity to an offspring only before birth,although IgA transferred to a neonate via breast milk is believed toplay a role in protecting the neonate against enteric infection.

The delivery of maternal IgG to the mammalian fetus and/or neonaterequires transport across an epithelial barrier which is largelyimpervious to macromolecules. The transport of macromolecules acrosssuch an epithelial barrier may occur by non-specific and specificreceptor-mediated mechanisms. Receptor non-specific mechanisms arerepresented by paracellular sieving events, the efficiency of which areinversely related to the molecular weight of the transported molecule.Transport of macromolecules such as IgG across this paracellular pathwayis highly inefficient.

Descriptions of receptor-mediated transport of immunoglobulins throughintestinal epithelial cells are limited thus far to the polymericimmunoglobulin receptor and the enterocyte receptor of IgG (a majorhistocompatibility complex (MHC) class I related Fc receptor). These tworeceptor systems differ in their specificity for immunoglobulin isotype,in their direction of immunoglobulin transport across the epithelialcell and in their tissue-specific expression. Both may play a role inmolding the immature immune system.

The polymeric immunoglobulin receptor is expressed on the basolateralsurfaces of enterocytes, hepatocytes and/or biliary duct epithelialcells. It transports polymeric IgA and IgM to the apical (luminal)surfaces, concentrating these immunoglobulins for antimicrobial defenseand antigen exclusion.

The enterocyte receptor for IgG, which has homology to the MHC class Iheavy chain and is associated with beta₂-microglobulin (β₂M), isexpressed on neonatal enterocytes of the rat and mouse. IgG istransported transcellularly in a luminal to serosal direction across theintestinal epithelium of these rodent neonates. On the apical surface ofthe enterocyte, the Fc portion of IgG is bound to the enterocytereceptor at the relatively acidic pH of the lumen (about pH 6.0).Following transcytosis to the basolateral plasma membrane, discharge ofthe immunoglobulin occurs at the relatively neutral pH of theinterstitial fluids (about pH 7.4). The rodent neonatal Fc receptor(FcRn) therefore could e responsible for delivery of maternal IgG to theneonate and as such may be responsible for the passive acquisition ofIgG during this period.

In humans, maternal IgG is actively transported across the placenta. Thereceptor responsible for this transport has been sought for many years.Several IgG-binding proteins have been isolated from placenta. FcγRIIwas detected in placental endothelium and FcγRIII insyncytiotrophoblasts. Both of these receptors, however, showed arelatively low affinity for monomeric IgG. Recently, the isolation fromplacenta of a cDNA encoding a human homolog of the rat and mouseenterocyte receptor for IgG was reported. (Story, C. M. et al., J. Exp.Med., Vol. 180:2377-2381, December 1994) The complete nucleotide anddeduced amino acid sequence is reported. This Fc receptor for IgG may beresponsible for the transport of maternal IgG to the human fetus (andeven possibly to the neonate), as the molecule is highly homologous overits open reading frame with the rat FcRn sequence (69% nucleotideidentity and 65% predicted amino acid identity). So called passiveimmunization in the human fetus (and possibly in the human neonate) nowmay become better understood.

In contrast to passive immunization which involves supplementing ahost's immune system with antibodies derived from another, activeimmunization involves stimulation of the host's own immune system togenerate in vivo the desired immune response. The most widely practicedmethods of active immunization in children and adults involve injectionsof an immunogen, once as an initial dose and then at least once again asa booster dose. These methods suffer many serious drawbacks, includingthe risks associated with the use of needles that can transmit diseasessuch as AIDS and hepatitis. (When tolerizing a patient against anallergen, the problems are compounded in that repeated injections over along period of time often are required.) These methods also do notnecessarily trigger adequately the first line of defense against manypathogens, that is, mucosal immunity. Mucous membranes line the airways,the reproductive system and the gastrointestinal tract, and this mucosalsurface represents the first portal of entry for many diseases. An oralvaccine that is easy to deliver and that triggers mucosal immunity wouldbe highly desirable.

Immunization using oral vaccines is problematic. Often little or noimmune response is achieved. To enhance the immune response, antigens ofinterest have been coupled to carriers that are known to be stronglyimmunogenic. For example, researchers have-delivered antigens usingBacille Calmette-Gurein (BCG) as a carrier; BCG is a bacteriumoriginally used as an oral vaccine against tuberculosis. A problem withsuch carriers is that the patient will develop antibodies against thecarrier itself, which can be troublesome if the carrier is used againfor delivering a different antigen to the same patient. To date, nogeneral strategy for oral vaccine: has proven successful.

Immunoglobulin and portions thereof in the past have been conjugated todrugs and imaging agents to target and destroy cell populations and toextend the half-lives of certain agents. Immunotoxins are an example ofsuch conjugates. Such conjugates, however, have never been proposed asuseful for initiating an immune response.

A small body of work has focused on the tolerogenic capacity ofimmunoglobulins coupled to oligonucleotides or proteins characteristicof autoimmune diseases. (See PCT WO 91/08773). This work is based uponthe notion that the induction of tolerance may be strongly influenced bycarrier moieties and that immunoglobulin carriers appear to be stronglytolerogenic. Isologous IgG is the preferred carrier, and intravenousadministration was the mode used for delivering the conjugates of IgG.Although this body of work extends for more than a decade, oraladministration is mentioned only once and only for conjugates where IgAis the immunoglobulin carrier. Thus, although tolerogenic immunoglobulinconjugates are known in the art, such conjugates have never beensuggested as agents for inducing a robust response against an antigencharacteristic of a pathogen. (To the contrary, the art suggests thatsuch conjugates, if anything, would tolerize a subject against apathogen which would be highly undesirable). In addition, it never hasbeen suggested that such conjugates would be effective tolerogens whenthe immunoglobulin is IgG and the mode of delivery is oral delivery.

3. SUMMARY OF THE INVENTION

The invention involves the discovery that antigens may be coupled tomolecules that bind to the FcRn receptor, such as immunoglobulins, orportions thereof, and delivered across epithelial barriers by activetransport through the enterocyte via FcRn receptors. The immunoglobulinor portion thereof binds to the FcRn receptor and acts as a carrier forthe antigen as the immunoglobulin or portion thereof is transportedacross the epithelial barrier by FcRn mediated-transport. The FcRnreceptor is present in the human epithelial tissue of children andadults, and the invention therefore permits effective strategies forimmunizing humans.

According to one aspect of the invention, a method for modulating theimmune system of a mammal is provided. An effective amount of aconjugate of an antigen and a FcRn binding partner is administered to anepithelial barrier of a mammal in need of such immune modulation. Theantigen is selected from the group consisting of: an antigen that ischaracteristic of a pathogen, an antigen that is characteristic of anautoimmune disease, an antigen that is characteristic of an allergen andan antigen that is characteristic of a tumor.

The FcRn binding partners of the present invention may be utilized forthe delivery of a wide variety of compounds and therapeutics andbioactive substances, including but not limited to, chemotherapy agentsfor the treatment of cancer, cytokines, including interferon; andhormones including insulin, human growth hormone (HGH), fertility drugs,calcitonin, calcitriol and other bioactive steroids. The FcRn bindingpartners of the present invention may further be utilized for thetargeted delivery of a delivery vehicle, such as liposomes.

In preferred embodiments, the FcRn binding partner is non-specific IgGor a FcRn binding fragment of IgG. Most preferably the FcRn bindingpartner is an Fc fragment of IgG. It also is preferred that the antigenbe covalently coupled to the FcRn binding partner. Preferably theconjugate is administered orally to the intestinal epithelium, in anaerosol to the lungs or intranasally. Such preparations may benonaseptic. Supplementary potentiating agents, as described below, maybe administered in addition.

In a preferred embodiment, when the compound to be delivered is apeptide or protein, the FcRn binding partner protein conjugate may besynthesized as a recombinant fusion protein. Examples of peptides andproteins which may be conjugated in this manner include cytokines;chemokines; growth factors, insulin, erythropoietin (EPO),neuropeptides, neuropeptide Y, neurotensin, transforming growth factorα, transforming growth factors β, interferon (IFN), and hormones.

The pharmaceutical compositions of the present invention relate to FcRnbinding partners conjugated to bioactive substances, including vaccinesor drugs for oral, sublingual or intranasal systemic delivery. Thepharmaceutical preparation of the present invention includes a conjugateof an antigen and a FcRn binding partner, wherein the antigen isselected from the group consisting of: an antigen that is characteristicof a pathogen, an antigen that is characteristic of an autoimmunedisease, an antigen that is characteristic of an allergen and an antigenthat is characteristic of a tumor. The pharmaceutical preparation of thepresent invention includes a conjugate of a drug or therapeutic and aFcRn binding partner. The preferred-FcRn binding partners are asdescribed above. The conjugate is present in an amount effective formodulating the immune response of a mammal. The pharmaceuticalpreparation of the present invention also includes a pharmaceuticallyacceptable carrier. When the antigen is characteristic of an autoimmunedisease or an allergen, then the pharmaceutical preparations of theinvention must be formulated in unit dosage form constructed andarranged for delivery to an epithelial carrier such as for oral deliveryto the intestinal epithelium, aerosol delivery to the pulmonaryepithelium and intranasal delivery to the nasal epithelium. Thus tabletscontaining IgG (or an FcRn binding portion thereof) coupled to any ofthe antigens as characterized above are embraced by the presentinvention.

The foregoing pharmaceutical preparations may be delivered together withsupplementary potentiating agents including adjuvants, cytokines,bioadhesives and the like. The supplementary potentiating agentsthemselves may be coupled to a FcRn binding partner to facilitate thedelivery of such agents across the epithelial barrier. Preferred modesof administration in general include oral dosages to the intestinalepithelium, aerosols to the lungs and intranasal dosages.

The present invention further relates to the synthesis, preparation anduse of the FcRn binding partner conjugates of the present invention as,or in, pharmaceutical compositions for oral and intranasal systemicdelivery of drugs and vaccines. The synthesis of the FcRn bindingpartner conjugates of the present invention comprises covalentlycoupling an antigen or a supplementary potentiating agent to an FcRnbinding partner, wherein the antigen or supplementary potentiating agentis selected as described above. The synthesis of the FcRn bindingpartner conjugates of the present invention alternatively comprisescovalently coupling a FcRn binding partner to a therapeutic or drug.Further, the synthesis of the FcRn binding partner conjugates of thepresent invention comprise covalently coupling a FcRn binding partner toa delivery vehicle, e.g. liposomes. The preferred FcRn binding partneralso is as described above. The conjugates then can be used to preparethe pharmaceutical preparations of the present invention.

In yet another aspect of the invention, the conjugate including theantigen crosses the epithelial barrier in an amount at least double theextent that the antigen crosses the epithelial barrier in anunconjugated form. It thus is an object of the invention to develop amechanism for increasing the ability of an antigen to cross anepithelial barrier.

Another object of the invention is to develop a new class of orallyactive immunogens and toleragens.

Another object of the invention is to develop improved methods forstimulating mucosal immunity.

These and other aspects of the invention are described in greater detailbelow.

4. BRIEF DESCRIPTION OF DRAWING

FIG. 1. Nucleotide and Amino Acid Sequence of Fc fragment of human IgG.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to FcRn binding partners modified for thetargeted delivery of vaccines, antigens, drugs, therapeutics andliposomes to epithelial barriers. The invention involves the discoverythat the human FcRn receptor is active in adult epithelial tissue andthe discovery that FcRn binding partners such as IgG or Fc fragments canbe used to transport other molecules, including antigens, acrossepithelial barriers. In this manner, “FcRn binding partners”, such asIgG or an FcRn binding portion thereof can be used to deliver an antigenor a therapeutic across an epithelial systemic circulation therebyeliciting a beneficial response or effect, e.g., an immune response.

The invention is useful whenever it is desirable to enhance the deliveryof an antigen across an epithelial barrier to the immune system. Theinvention thus may be used to deliver antigens across intestinalepithelial tissue, lung epithelial tissue and other mucosal surfacesincluding nasal surfaces, vaginal surfaces, colon surfaces and binarytree surfaces. The invention may be used to modulate a subject's immunesystem such as by stimulating a humoral antibody response against anantigen, by stimulating T cell activity, or by stimulating tolerance toan antigen. As used herein, subject means: humans, primates, horses,cows, sheep, pigs goats, dogs, cats, chickens and rodents. Whendelivering, tumor antigens, the invention may be used to treat subjectshaving disease amenable to immunity mediated rejection, such asnon-solid tumors or solid tumors of small size. It is also contemplatedthat delivery of tumor antigens by the methods described herein will beuseful for treatment subsequent to removal of large solid tumors. Theinvention may also be used to treat subjects who are suspected of havingcancer.

The invention is also useful whenever it is desirable to achievesystemic delivery of a therapeutic or drug or delivery vehicle orprotein or combinations thereof, across an epithelial barrier tosystemic circulation. The invention, thus may be used to delivertherapeutics across intestinal epithelial tissue, lung epithelialtissue, and other mucosal epithelial surfaces including nasal surfaces,vaginal surfaces, colon and rectal surfaces and binary free surfaces.The invention may be used to administer a therapeutic to elicit abeneficial effect. The FcRn binding partner conjugates are designed todeliver a wide variety of therapeutics including RNA and DNA nucleotidesas used, for example, in gene therapy, peptides, carbohydrates and smallmolecules. These therapeutics include but are not limited to, anticancerand chemotherapeutic drugs, e.g., doxorubicin; anti-inflammatory drugs,e.g., steroids; drugs for the treatment of cardiovascular disease, e.g.,cholinesterase inhibitors; drugs for the treatment of disorders relatedto viral infection, e.g. hepatic cirrhosis resulting from hepatitisinfection; drugs for the treatment of weight disorders, e.g.,amphetamines; antibacterial agents, antifungal agents, cytokines,fertility drugs, antibiotics, hormones, steroids, etc.

The invention involves the formation of a conjugate of an FcRn bindingpartner and an antigen or a therapeutic or drug. By conjugate it ismeant two or more entities bound to one another by any physiochemicalmeans, including, but not limited to, hydrophobic interaction, covalentinteraction, hydrogen bond interaction, or ionic interaction between abioactive substance, such as, an antigen or a therapeutic and thenon-specific hydrophobic portions of an antibody molecule,antibody-antigen specific binding and covalent coupling. The nature ofthe preferred bonding will depend, among other things, upon the mode ofadministration and the pharmaceutical carriers used to deliver theconjugate to the selected epithelial barrier. For example, some bondsare not as well suited as others to withstand certain environments suchas the stomach, but can be protected therefrom by delivery systems whichbypass the stomach. It, of course, is important that the bond betweenthe FcRn binding partner and the antigen be of such a nature that itdoes not destroy the ability of the FcRn binding partner to bind to theFcRn receptor. Such bonds are well known to those of ordinary skill inthe art examples are provided in greater detail below. The conjugatefurther may be formed as a fusion protein, also discussed in greaterdetail below.

5.1. FcRn Binding Partners

An FcRn binding partner means any entity that can be specifically boundby the FcRn receptor with consequent active transport by the FcRnreceptor of the FcRn binding partner. As mentioned above, the FcRnreceptor has been isolated for several mammalian species, includinghumans. The sequence of the human FcRn, rat FcRn and mouse FcRn may befound in Story, C. M. et al, J. Exp. Med., vol. 180:2377-2381, December1994. The FcRn receptor molecule now is well characterized. The FcRnreceptor binds IgG (but not other immunoglobulin classes such as IgA,IgD, IgM and IgE) at a relatively lower pH, actively transports the IgGtranscellularly in a luminal to serosal direction, and then releases theIgG at a relatively higher pH found in the interstitial fluids. As willbe recognized by those of ordinary skill in the art, FcRn receptors canbe isolated by cloning or by affinity purification using, for example,monoclonal antibodies. Such isolated FcRn receptors then can be used toidentify and isolate FcRn binding partners, as described below.

FcRn binding partners of the present invention encompass any entity thatcan be specifically bound by the FcRn receptor, including whole IgG, theFc fragment of IgG and other fragments of IgG that include the completebinding region for the FcRn receptor. The region of the Fc portion ofIgG that binds to the FcRn receptor has been described based upon X-raycrystallography (Burmaister, W. P. et al., Nature, 1994; 372:379-378.)The major contact area of Fc with the FcRn receptor is near the junctionof the C_(H)2 and C_(H)3 domains. Potential contacts are residues 248,250-257, 272, 285, 288, 290-291, 308-311 and 314 in CH_(H)2 and 385-387,428 and 433-436 in C_(H)3. (These sites are distinct from thoseidentified by subclass comparison or by site-directed mutagenesis asimportant for Fc binding to leukocyte FcγRI and FcγRII.) The foregoingFc-FcRn contacts are all within a single Ig heavy chain. It has beennoted previously that two FcRn receptors can bind a single Fc molecule.The crystallographic data suggest that in such a complex, each FcRnmolecule binds a single polypeptide of the Fc homodimer.

In an embodiment of the present invention, FcRn binding partners otherthan whole IgG may be used to transport therapeutics across theepithelial barrier. In such an embodiment, it is preferred that a FcRnbinding partner is chosen which binds the FcRn with higher affinity thanwhole IgG. Such a FcRn binding partner has utility in utilizing the FcRnto achieve active transport of a conjugated therapeutic across theepithelial barrier, and also has utility to prevent the binding andtransport of whole IgG across the epithelial barrier via the FcRn. TheFcRn binding activity of these higher affinity FcRn binding partners maybe measured using standard assays known to those skilled in the art,including: (a) transport assays using polarized cells that naturallyexpress the FcRn, or have been genetically engineered to express theFcRn or the alpha chain of the FcRn; (b) protein:protein binding assaysusing soluble FcRn or fragments thereof, or immobilized FcRn; (c)binding assays utilizing polarized or non-polarized cells that naturallyexpress the FcRn, or have been genetically engineered to express theFcRn or the alpha chain of the FcRn.

5.1.1. Recombinant Synthesis of FcRn Binding Partners

In accordance with the present invention, the FcRn binding partner maybe produced by recombinant genetic engineering techniques. Within thescope of the invention are nucleotide sequences encoding human FcRnbinding partners. The FcRn binding partners include whole IgG, the Fcfragment of IgG and other fragments of IgG that include the completebinding region for the FcRn. The major contact sites include amino acidresidues 248, 250-257, 272, 285, 288, 290-291, 308-311 and 314 of theC_(H)2 and amino acid residues 385-387, 428 and 433-436 of the C_(H)3.Therefore in a preferred embodiment of the present invention arenucleotide sequences encoding regions of the IgG Fc spanning these aminoacid residues.

Given the foregoing information, those of ordinary skill in the art willreadily recognize that the Fc region of IgG can be modified according towell-recognized procedures such as site-directed mutagenesis and thelike to yield modified IgG or modified Fc fragments or portions thereofthat will be bound by the FcRn receptor. Such modifications includemodifications remote from the FcRn contact sites as well asmodifications within the contact sites that preserve or even enhancebinding. In addition, other binding partners can be identified andisolated. Antibodies or portions thereof specific for the FcRn receptorand capable of being transported by FcRn once bound can be identifiedand isolated using well established techniques. Likewise, randomlygenerated molecularly diverse libraries can be screened and moleculesthat are bound and transported by FcRn receptors can be isolated usingconventional techniques. It is not intended that the invention belimited by the selection of any particular FcRn binding partner.

If the peptide is composed entirely of gene-encoded amino acids, or aportion of it is so composed, the peptide or the relevant portion mayalso be synthesized using conventional recombinant genetic engineeringtechniques.

For recombinant production, a polynucleotide sequence encoding the FcRnbinding partner is inserted into an appropriate expression vehicle,i.e., a vector which contains the necessary elements for thetranscription and translation of the inserted coding sequence, or in thecase of an RNA viral vector, the necessary elements for replication andtranslation. The expression vehicle is then transfected into a suitabletarget cell which will express the peptide. Depending on the expressionsystem used, the expressed peptide is then isolated by procedureswell-established in the art. Methods for recombinant protein and peptideproduction are well known in the art (see, e.g., Maniatis et al., 1989,Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory,N.Y.; and Ausubel et al., 1989, Current Protocols in Molecular Biology,Greene Publishing Associates and Wiley Interscience, N.Y.).

To increase efficiency of production, the polynucleotide can be designedto encode multiple units of the FcRn binding partner separated byenzymatic cleavage sites. The resulting polypeptide can be cleaved(e.g., by treatment with the appropriate enzyme) in order to recover thepeptide units. This can increase the yield of peptides driven by asingle promoter. When used in appropriate viral expression systems, thetranslation of each peptide encoded by the mRNA is directed internallyin the transcript; e.g., by an internal ribosome entry site, IRES. Thus,the polycistronic construct directs the transcription of a single, largepolycistronic mRNA which, in turn, directs the translation of multiple,individual peptides. This approach eliminates the production andenzymatic processing of polyproteins and may significantly increaseyield of peptide driven by a single promoter.

A variety of host-expression vector systems may be utilized to expressthe FcRn binding partners described herein. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage DNA or plasmid DNA expression vectors containing anappropriate coding sequence; yeast or filamentous fungi transformed withrecombinant yeast or fungi expression vectors containing an appropriatecoding sequence; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing an appropriate codingsequence; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing an appropriate coding sequence; or animal cellsystems.

The expression elements of the expression systems vary in their strengthand specificities. Depending on the host/vector system utilized, any ofa number of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used in the expressionvector. For example, when cloning in bacterial systems, induciblepromoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lachybrid promoter) and the like may be used; when cloning in insect cellsystems, promoters such as the baculovirus polyhedron promoter may beused; when cloning in plant cell systems, promoters derived from thegenome of plant cells (e.g., heat shock promoters; the promoter for thesmall subunit of RUBISCO; the promoter for the chlorophyll a/b bindingprotein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; thecoat protein promoter of TMV) may be used; when cloning in mammaliancell systems, promoters derived from the genome of mammalian cells(e.g., metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5 K promoter) may beused; when generating cell lines that contain multiple copies ofexpression product, SV40-, BPV- and EBV-based vectors may be used withan appropriate selectable marker.

In cases where plant expression vectors are used, the expression ofsequences encoding linear or non-cyclized forms of the cyclic peptidesof the invention may be driven by any of a number of promoters. Forexample, viral promoters such as the 35S RNA and 19S RNA promoters ofCaMV (Brisson et al., 1984, Nature 310:511-514), or the coat proteinpromoter of TMV (Takamatsu et al., 1987, EMBO J. 6:307-311) may be used;alternatively, plant promoters such as the small subunit of RUBISCO(Coruzzi et al., 1984, EMBO J. 3:1671-1680; Broglie et al., 1984,Science 224:838-843) or heat shock promoters, e.g., soybean hsp17.5-E orhsp17.3-B (Gurley et al., 1986, Mol. Cell. Biol. 6:559-565) may be used.These constructs can be introduced into plant cells using Ti plasmids,Ri plasmids, plant virus vectors, direct DNA transformation,microinjection, electroporation, etc. For reviews of such techniquessee, e.g., Weissbach & Weissbach, 1988, Methods for Plant MolecularBiology, Academic Press, NY, Section VIII, pp. 421-463; and Grierson &Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.

In one insect expression system that may be used to produce the peptidesof the invention, Autographa californica nuclear polyhidrosis virus(AcNPV) is used as a vector to express the foreign genes. The virusgrows in Spodoptera frugiperda cells. A coding sequence may be clonedinto non-essential regions (for example the polyhedron gene) of thevirus and placed under control of an AcNPV promoter (for example, thepolyhedron promoter). Successful insertion of a coding sequence willresult in inactivation of the polyhedron gene and production ofnon-occluded recombinant virus (i.e., virus lacking the proteinadeouscoat coded for by the polyhedron gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed. (e.g., see Smith et al., 1983, J. Virol. 46:584;Smith, U.S. Pat. No. 4,215,051). Further examples of this expressionsystem may be found in Current Protocols in Molecular Biology, Vol. 2,Ausubel et al., eds., Greene Publish. Assoc. & Wiley Interscience.

In mammalian host cells, a number of viral based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, a coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingpeptide in infected hosts. (e.g., See Logan &. Shenk, 1984, Proc. Natl.Acad. Sci. (USA) 81:3655-3659). Alternatively, the vaccinia 7.5 Kpromoter may be used, (see, e.g., Mackett et al., 1982, Proc. Natl.Acad. Sci. (USA) 79:7415-7419; Mackett et al., 1984, J. Virol.49:857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. 79:4927-4931).

5.2. FcRn Binding Partners Conjugated to Antigens for Vaccine Delivery

The FcRn binding partner is conjugated with an antigen. An antigen asused herein falls into four classes: 1) antigens that are characteristicof a pathogen; 2) antigens that are characteristic of an autoimmunedisease; 3) antigens that are characteristic of an allergen; and 4)antigens that are characteristic of a tumor. Antigens in general includepolysaccharides, glycolipids, glycoproteins, peptides, proteins,carbohydrates and lipids from cell surfaces, cytoplasm, nuclei,mitochondria and the like.

Antigens that are characteristic of pathogens include antigens derivedfrom viruses, bacteria, parasites or fungi. Examples of importantpathogens include vibrio choleras, enterotoxigenic Escherichia coli,rotavirus, Clostridium difficile, Shigella species, Salmonella typhi,parainfluenza virus, influenza virus, Streptococcus pneumonias, Borellaburgdorferi, HIV, Streptococcus mutans, Plasmodium falciparum,Staphylococcus aureus, rabies virus and Epstein-Barr virus.

Viruses in general include but are not limited to those in the followingfamilies: picornaviridae; caliciviridae; togaviridae; flaviviridae;coronaviridae; rhabdoviridae; filoviridae; paramyxoviridae;orthomyxoviridae; bunyaviridae; arenaviridae; reoviridae; retroviridae;hepadnaviridae; parvoviridae; papovaviridae; adenoviridae;herpesviridae; and poxyviridae.

Bacteria in general include but are not limited to: P. aeruginosa; E.coli, Klebsiella sp.; Serratia sp.; Pseudomanas sp.; P. cepacia;Acinetobacter sp.; S. epidermis; E. faecalis; S. pneumonias; S. aureus;Haemophilus sp.; Neisseria Sp.; N. meningitidis; Bacteroides sp.;Citrobacter sp.; Branhamella sp.; Salmonelia sp.; Shigella sp.; S.pyogenes; Proteus sp.; Clostridium sp.; Erysipelothrix sp.; Lesteriasp.; Pasteurella multocida; Streptobacillus sp.; Spirillum sp.;Fusospirocheta sp.; Treponema pallidum; Borrelia sp.; Actinomycetes;Mycoplasma sp.; Chlamydia sp.; Rickettsia sp.; Spirochaeta; Legionellasp.; Mycobacteria sp.; Ureaplasma sp.; Streptomyces sp.; Trichomorassp.; and P. mirabilis.

Parasites include but are not limited to: Plasmodium falciparum, P.vivax, P. ovale, P. malaria; Toxoplasma gondii; Leishmania mexicana, L.tropica, L. major, L. aethiopica, L. donovani, Trypanosoma cruzi, T.brucei, Schistosoma mansoni, S. haematobium, S. japonium; Trichinellaspiralis; Wuchereria bancrofti; Brugia malayli; Entamoeba histolytica;Enterobius vermiculoarus; Taenia solium, T. saginata, Trichomonasvaginatis, T. hominis, T. tenax; Giardia lamblia; Cryptosporidiumparvum; Pneumocytis carinii, Babesia bovis, B. divergens, B. microti,Isospore belli, L hominis; Dientamoeba fragiles; Onchocerca volvulus;Ascaris lumbricoides, Necator americanis; Ancylostoma duodenale;Strongyloides stercoralis; Capillaria philippinensis; Angiostrongyluscantonensis; Hymenolepis nana; Diphyllobothrium latum; Echinococcusgranulosus, E. multilocularis; Paragonimus westermani, P. caliensis;Chlonorchis sinensis; Opisthorchis felineas, G. Viverini, Fasciolahepatica Sarcoptes scabiei, Pediculus humanus; Phthirius pubis; andDermatobia hominis.

Fungi in general include but are not limited to: Cryptococcusneoformans; Blastomyces dermatitidis; Aiellomyces dermatitidis;Histoplasfria capsulatum; Coccidioides immitis; Candids species,including C. albicans, C. tropicalis, C. parapsilosis, C. guilliermondiiand C. krusei, Aspergillus species, including A. fumigatus, A. flavusand A. niger, Rhizopus species; Rhizomucor species; Cunninghammellaspecies; Apophysomyces species, including A. saksenaea, A. mucor and A.absidia; Sporothrix schenckii, Paracoccidioides brasiliensis;Pseudallescheria boydii, Torulopsis glabrata; and Dermatophyres species.

Antigens that are characteristic of autoimmune disease typically will bederived from the cell surface, cytoplasm, nucleus, mitochondria and thelike of mammalian tissues. Examples include antigens characteristic ofuveitis (e.g. S antigen), diabetes mellitus, multiple sclerosis,systemic lupus erythematosus, Hashimoto's thyroiditis, myastheniagravis, primary myxoedema, thyrotoxicosis, rheumatoid arthritis,pernicious anemia, Addison's disease, scleroderma, autoimmune atrophicgastritis, premature menopause (few cases), male infertility (fewcases), juvenile diabetes, Goodpasture's syndrome, pemphigus vulgaris,pemphigoid, sympathetic opthalmia, phacogenic uveitis, autoimmunehaemolytic anemia, idiopathic thrombocylopenic purpura, idiopathicfeucopenia, pr imary biliary cirrhosis (few cases), ulcerative colitis,Siogren's syndrome, Wegener's granulomatosis, poly/dermatomyositis, anddiscold lupus erythromatosus.

Antigens that are allergens are generally proteins or glycoproteins,although allergens may also be low molecular weight allergenic haptensthat induce allergy after covalently combining with a protein carrier(Remington's Pharmaceutical Sciences). Allergens include antigensderived from pollens, dust, molds, spores, dander, insects and foods.Specific examples include. the urushiols (pentadecylcatechol orheptadecyicatechol) of Toxicodendron species such as poison ivy, poisonoak and poison sumac, and the sesquiterpenoid lactones of ragweed andrelated plants.

Antigens that are characteristic of tumor antigens typically will bederived from the cell surface, cytoplasm, nucleus, organelles and thelike of cells of tumor tissue. Examples include antigens characteristicof tumor proteins, including proteins encoded by mutated oncogenes;viral proteins associated with tumors; and tumor mucins and glycolipids.Tumors include, but are not limited to, those from the following sitesof cancer and types of cancer: lip, nasopharynx, pharynx and oralcavity, esophagus, stomach, colon, rectum, liver, gall bladder, binarytree, pancreas, larynx, lung and bronchus, melanoma of skin, breast,cervix, uteri, uterus, ovary, bladder, kidney, brain and other parts ofthe nervous system, thyroid, prostate, testes, Hodgkin's disease,non-Hodgkin's lymphoma, multiple myeloma and leukemia. Viral proteinsassociated with tumors would be those from the classes of viruses notedabove. Antigens characteristic of tumors may be proteins not usuallyexpressed by a tumor precursor cell, or may be a protein which isnormally expressed in a tumor precursor cell, but having a mutationcharacteristic of a tumor. An antigen characteristic of a tumor may be amutant variant of the normal protein-having an altered activity orsubcellular distribution. Mutations of genes giving rise to tumorantigens, in addition to those specified above, may be in the codingregion, 5′ or 3′ noncoding regions, or introns of a gene, and may be theresult of point mutations frameshifts, deletions, additions,duplications, chromosomal rearrangements and the like. One of ordinaryskill in the art is familiar with the broad variety of alterations tonormal gene structure and expression which gives rise to tumor antigens.Specific examples of tumor antigens include: proteins such asIg-idiotype of B cell lymphoma, mutant cyclin-dependent kinase 4ofmelanoma, Pmel-17 (gp 100) of melanoma, MART-1 (Melan-A) of melanoma,p15 protein of melanoma, tyrosinase of melanoma, MAGE 1, 2 and 3 ofmelanoma, thyroid medullary, small cell lung cancer, colon and/orbronchial squamous cell cancer, BAGE of bladder, melanoma, breast, andsquamous-cell carcinoma, gp75 of melanoma, oncofetal antigen ofmelanoma; carbohydrate/lipids such as muci mucin of breast, pancreas,and ovarian cancer, GM2 and GD2 gangliosides of melanoma; oncogenes suchas mutant p53 of carcinoma, mutant ras of colon cancer and HER21neuproto-onco-gene of breast carcinoma; viral products such as humanpapilloma virus proteins of squamous cell cancers of cervix andesophagus. It is also contemplated that proteinaceous tumor antigens maybe presented by HLA molecules as specific peptides derived from thewhole protein. Metabolic processing of proteins to yield antigenicpeptides is well known in the art; for example see U.S. Pat. No.5,342,774 (Boon et al.). The present method thus encompasses delivery ofantigenic peptides and such peptides in a larger polypeptide or wholeprotein which give rise to antigenic peptides. Delivery of antigenicpeptides or proteins may give rise to humoral or cellular immunity.

Generally, subjects can receive an effective amount of the tumorantigen, and/or peptide derived therefrom by one or more of the methodsdetailed below. Initial doses can be followed by booster doses,following immunization protocols standard in the art. Delivery of tumorantigens thus may stimulate proliferation of cytolytic T lymphocytes.

In the cases of protein and peptide antigens, covalent linking to anFcRn partner is intended to include linkage by peptide bonds in a singlepqlypeptide chain. Established methods (Sambrook et al., MolecularCloning: A Laboratory Manual. Cold Spring Harbor Press, Cold SpringHarbor, N.Y. 1989) would be used to engineer DNA encoding a fusionprotein comprised of the antigenic peptide or protein and an FcRnpartner. This DNA would be placed in an expression vector and introducedinto bacterial or eukaryotic cells by established methods. The fusionprotein would be purified from the cells or from the culture medium byestablished methods.

In accordance with the present invention, a conjugate may be formed as afusion protein. Conjugates include fusions of the FcRn binding partnerto a protein, peptide or protein derivative such as those listed hereinincluding, but not limited to, antigens, allergens, pathogens or toother proteins or protein derivatives of potential therapeutic interestsuch as growth factors, colony stimulating factors, growth inhibitoryfactors, signaling molecules, hormones, steroids, neurotransmitters, ormorphogens that would be of use when delivered across an epithelialbarrier.

By way of example, but not limitation, proteins used in fusion proteinsto synthesize conjugates may include Platelet Derived Growth Factor(PDGF) (U.S. Pat. No. 4,766,073), Platelet Derived Endothelial CellGrowth Factor (PD-ECGF) (U.S. Pat. No. 5,227,302), Human pituitaryGrowth Hormone (HGH) (U.S. Pat. No. 3,853,833), Transforming GrowthFactor Beta (TGFβ) (U.S. Pat. No. 5,168,051), Transforming Growth FactorAlpha (TGFα) (U.S. Pat. No. 5,633,147), Keratinocyte Growth Factor (KGF)(U.S. Pat. No. 5,731,170), Insulin-like Growth Factor I (IGF-I) (U.S.Pat. No. 4,963,665), Epidermal Growth Factor (EGF) (U.S. Pat. No.5,096,825), Erythropoietin (EPO) (U.S. Pat. No. 4,703,008),Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) (U.S. Pat. No.5,200,327), M-CSF (U.S. Pat. No. 5,171,675), Colony Stimulating Factor-1(CSF-1) (U.S. Pat. No. 4,847,201), Steel factor, Calcitonin, AP-1proteins (U.S. Pat. No. 5,238,839), Brain Derived Neurotrophic Factor(BDNF) (U.S. Pat. No. 5,229,500). All of the references cited above areincorporated herein by reference in their entirety.

In one embodiment, fusion proteins of the present invention areconstructed in which the conjugate consists of an Fc fragment (startingwith the amino acids D-K-T-H at the N-terminus of the CH1 region,including the hinge and CH2 regions, and continuing through the S-P-G-Ksequence in CH3 region) fused to one of the above listed proteins. As anexample, the nucleotide sequences encoding Erythropoietin (EPO.) arefused to the Fc binding partner.

In order to engineer the DNA vector encoding the fusion protein FcRnbinding partner conjugate, the mammalian expression vector scFvE-sec(described in Persic, et.al. GENE 187:1-8 1997 and incorporated hereinby reference in its entirety) was linearized with BssH2 and Not1 and thelinearized plasmid was gel purified by protocols standard in the art.

The DNA encoding the protein to be fused to the Fc was prepared asfollows. The entire (mouse) EPO sequence was removed from the expressionvector (described in Bill et al., Biochem. Biophy. Acta 1261:35-43,1995, incorporated herein by reference in its entirety) by therestriction enzymes BamH1 and Sal1 sites outside the EPO insert. Thelinearized fragment was gel purified and modified by standard PCR(Polymerase Chain Reaction) protocols and a BssH2 site added adjacent tothe DNA encoding the first N-terminal alanine of mouse EPO (5′ end); theDNA encoding the C-terminal end of mouse EPO was modified with asequence corresponding to an ala-ala-ala linker followed by a Sal1restriction site (3′ end). The EPO fragment was then cut with BssH1 andSal1 and gel purified by standard protocols in the art. The Fc fragmentwas cut with restriction enzymes of SalI and NotI and gel purified.

The three linearized and gel purified DNAs described above, includingthe scfve-sec vector, Fc fragment and modified EPO DNAs were ligatedinto a single circular vector by a triple ligation using standardprotocols in the art.

It is noted that the DNA encoding EPO may be replaced with DNA encodingany of the above proteins or other proteins of interest or proteinfragments thereof by standard techniques to create an Fc fusion proteinleading to a conjugate. Similarly this invention allows for thereplacement of DNA encoding the Fc fragment in the vector with DNAencoding a FcRn binding partner as described herein.

Additionally, this invention allows for the replacement of both the DNAencoding EPO and the DNA encoding the Fc fragment such that the finalvector results in a conjugate that associates with the FcRn.

In order to demonstrate that a conjugate made by the fusion of a FcRnbinding partner and a protein of interest is capable of retainingbiological activity, the example protein above was expressed and assayedfor biological activity of erythropoietin in the following manner. Themammalian expression vector containing the EPO-Fc fusion was transfectedinto CHO (Chinese Hamster Ovary) cells and expressed by standardprotocols in the art. Supernatants of transfected or non-transfected CHOcells were collected and injected subcutaneously into Balbc mice.Reticulocyte counts of mice were obtained by coulter FACs analysis bytechniques known in the field of the art. Results demonstrated that miceinjected with the supernatants of the transfected cells had reticulocyte(mature erythrocytes) counts several fold higher than mice injected withcontrol (untransfected supernatants). Since EPO has been documented tostimulate the production of erythrocytes, the results disclosed hereinsupport the ability of the invention to synthesize biologically activeFcRn binding partner conjugates.

Similarly, fusion proteins substituting the Fc fragment for an alternateFcRn binding partner domain in the vector described above would beexpected to retain biological activity.

5.3. FcRn Binding Partners Conjugated to Therapeutics for Systemic DrugDelivery

The FcRn binding partners may be conjugated to a variety of therapeuticsor drugs for targeted systemic delivery. The present inventionencompasses the targeted systemic delivery of biologically activesubstances.

As used herein, the term “biologically active substance” prefers toeukaryotic and procaryotic cells, viruses, vectors, proteins, peptides,nucleic acids, polysaccharides and carbohydrates, lipids, glycoproteins,and combinations thereof, and synthetic organic and inorganic drugsexerting a biological effect when administered to an animal. For ease ofreference, the term is also used to include detectable compounds such asradiopaque compounds including air and barium, magnetic compounds. Theactive substance can be soluble or insoluble in water. Examples ofbiologically active substances include anti-angiogenesis factors,antibodies, growth factors hormones, enzymes, and drugs such assteroids, anti-cancer drugs or antibiotics.

In diagnostic embodiments, the FcRn binding partners may also beconjugated to a pharmaceutically acceptable gamma-emitting moiety,including but not limited to, indium and technetium, magnetic particles,radiopaque materials such as air or barium and fluorescent compounds.

By way of example, and not be limitation, the following classes of drugsmay be conjugated to FcRn binding partners for the purposes of deliveryto epithelial borders:

Antineoplastic Compounds. Nitrosoureas, e.g., carmustine, lomustine,semustine, strepzotocin; Methylhydrazines, e.g., procarbazine,dacarbazine; steroid hormones, e.g., glucocorticoids, estrogens,progestins, androgens, tetrahydrodesoxycaricosterone, cytokines andgrowth factors; Asparaginase.

Immunoactive Compounds. Immunosuppressives, e.g., pyrimethamine,trimethopterin, penicillamine, cyclosporine, azathioprine;immunostimulants, e.g., levamisole, diethyl dithiocarbamate,enkephalins, endorphins.

Antimicrobial Compounds. Antibiotics, e.g., β lactam, penicillin,cephalosporins, carbapenims and monobactams, β-lactamase inhibitors,aminoglycosides, macrolides, tetracyclins, spectinomycin; Antimalarials,Amebicides, Antiprotazoal, Antifungals, e.g., amphotericin β, Antiviral,e.g., acyclovir, idoxuridine, ribavirin, trifluridine, vidarbine,gancyclovir.

Parasiticides. Antihalmintics, Radiopharmaceutics, gastrointestinaldrugs.

Hematologic Compounds. Immunoglobulins; blood clotting proteins; e.g.,antihemophilic factor, factor IX complex; anticoagulants, e.g.,dicumarol, heparin Na; fibrolysin inhibitors, tranexamic acid.

Cardiovascular Drugs. Peripheral antiadrenergic drugs, centrally actingantihypertensive drugs, e.g., methyldopa, methyldopa HCl;antihypertensive direct vasodilators, e.g., diazoxide, hydralazine HCl;drugs affecting renin-angiotensin system; peripheral vasodilators,phentolamine; antianginal drugs; cardiac glycosides; inodilators; e.g.,amrinone, milrinone, enoximone, fenoximone, imazodan, sulmazole;antidysrhythmic; calcium entry blockers; drugs affecting blood lipids;ranitidine, bosentan, rezulin.

Respiratory Drugs. Sypathomimetic drugs: albuterol, bitolterol mesylate,dobutamine HCl, dopamine HCl, ephedrine SO, epinephrine, fenfluramineHCl, isoproterenol HCl, methoxamine HCl, norepinephrine bitartrate,phenylephrine HCl, ritodrine HCl; cholinomimetic drugs, e.g.,acetylcholine Cl; anticholinesterases, e.g., edrophonium Cl;cholinesterase reactivators; adrenergic blocking drugs, e.g., acebutololHCl, atenolol, esmolol HCl, labetalol HCl, metoprolol, nadolol,phentolamine mesylate, propanolol HCl; antimuscarinic drugs, e.g.,anisotropine methylbromide, atropine SO4, clinidium Br, glycopyrrolate,ipratropium Br, scopolamine HBr.

Neuromuscular Blocking Drugs. Depolarizing, e.g., atracurium besylate,hexafluorenium Br, metocurine iodide, succinylcholine Cl, tubocuracrineCl, vecuronium Br; centrally acting muscle relaxants, e.g., baclofen.

Neurotransmitters and neurotransmitter Agents. Acetylcholine, adenosine,adenosine triphosphate, amino acid neurotransmitters, e.g., excitatoryamino acids, GABA, glycine; biogenic amine neurotransmitters, e.g.,dopamine, epinephrine, histamine, norepinephrine, octopamine, serotonin,tyramine; neuropeptides, nitric oxide, K+ channel toxins,

Antiparkinson Drugs. amaltidine HCl, benztropine mesylate, e.g.,carbidopa.

Diuretic Drugs. Dichlorphenamide, methazolamide, bendroflumethiazide,polythiazide.

Uterine, Antimigraine Drugs. Carboprost tromethamine, mesylate,methysergide maleate.

Hormones. Pituitary hormones, e.g., chorionic gonadotropin, cosyntropin,menotropins, somatotropin, iorticotropin, protirelin, thyrotropin,vasopressin, lypressin; adrenal hormones, e.g., beclomethasonedipropionate, betamethasone, dexamethasone, triamcinolone; pancreatichormones, e.g., glucagon, insulin; parathyroid hormone, e.g.,dihydrochysterol; thyroid hormones, e.g., calcitonin etidronatedisodium, levothyroxine Na, liothyronine Na, liotrix, thyroglobulin,teriparatide acetate; antithyroid drugs; estrogenic hormones; progestinsand antagonists, hormonal contraceptives, testicular hormones;gastrointestinal hormones: cholecystokinin, enteroglycan, galanin,gastric inhibitory polypeptide, epidermal growth factor-urogastrone,gastric inhibitory polypeptide, gastrin-releasing peptide, gastrins,pentagastrin, tetragastrin, motilin, peptide YY, secretin, vasoactiveintestinal peptide, sincalide.

Enzymes. Hyaluronidase, streptokinase, tissue plasminogen activator,urokinase, PGE-adenosine deaminase.

Intravenous Anesthetics. Droperidol, etomidate, fetanylcitrate/droperidol, hexobarbital, ketamine HCl, methohexital Na,thiamylal Na, thiopental Na.

Antiepileptics. Carbamazepine, clonazepam, divalproex Na, ethosuximide,mephenytoin, paramethadione, phenytoin, primidone.

Peptides and proteins. The FcRn binding partners may be conjugated topeptides or polypeptides, e.g., ankyrins, arrestins, bacterial membraneproteins, clathrin, connexins, dystrophin, endothelin receptor,spectrin, selectin, cytokines; chemokines; growth factors, insulin,erythropoietin (EPO), tumor necrosis factor (TNF), neuropeptides,neuropeptide Y, neurotensin, transforming growth factor α, transforminggrowth factor β, interferon (IFN), and hormones, growth inhibitors, e.g.genistein, steroids etc; glycoproteins, e.g., ABC transporters, plateletglycoproteins, GPIb-IX complex, GPIIb-IIIa complex, vitronectin,thrombomodulin, CD4, CD55, CD58, CD59, CD44, lymphocyefunction-associated antigen, intercellular adhesion molecule, vascularcell adhesion molecule, Thy-1, antiporters, CA-15-3 antigen,fibronectins, laminin, myelin-associated glycoprotein, GAP, GAP-43. Inthis embodiment of the present invention, the polypeptide therapeuticsmay be covalently conjugated to the FcRn binding partner or the FcRnbinding partner and therapeutic may be expressed as a fusion proteinusing standard recombinant genetic techniques, see Section 5.1.1 infra.

Cytokines and Cytokine Receptors. Examples of cytokines and receptorsthereof which may be delivered via a FcRn binding partner or conjugatedto an FcRn binding partner in accordance with the present invention,include, but are not limited to: Interleukin-1 (IL-1), IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-4receptor, IL-5 receptor, IL-6 receptor, IL-7 receptor, IL-8 receptor,IL-9 receptor, IL-10 receptor, IL-11 receptor, IL-12 receptor, IL-13receptor, IL-14 receptor, IL-15 receptor, IL-16 receptor, IL-17receptor, IL-18 receptor, lymphokine inhibitory factor, macrophagecolony stimulating factor, platelet derived growth factor, stem cellfactor, tumor growth factor β, tumor necrosis factor, lymphotoxin, Fas,granulocyte colony stimulating factor, granulocyte macrophage colonystimulating factor, interferon α, interferon β, interferon γ.

Growth Factors and Protein Hormones. Examples of growth factors andreceptors thereof and protein hormones and receptors thereof which maybe delivered via a FcRn binding partner or conjugated to an FcRn bindingpartner in accordance with the present invention, include, but are notlimited to: erythropoietin, angiogenin, hepatocyte growth factor,fibroblast growth factor, keratinocyte growth factor, nerve growthfactor, tumor growth factor α, thrombopoietin, thyroid stimulatingfactor, thyroid releasing hormone, neurotrophin, epidermal growthfactor, VEGF, ciliary neurotrophic factor, LDL, sqmatomedin, insulingrowth factor, insulin-like growth factor I and II.

Chemokines. Examples of chemokines and receptors thereof which may bedelivered via a FcRn binding parnter or conjugated to an FcRn bindingpartner in accordance with the present invention, include, but are notlimited to: ENA-78, ELC, GRO-α, GRO-β, GRO-γ, HRG, LIF, IP-10, MCP-1,MCP-2, MCP-3, MCP-4, MIP-1α, MIP-1β, MIG, MDC, NT-3, NT-4, SCF, LIF,leptin, RANTES, lymphotactin, eotaxin-1, eotaxin-2, TARC, TECK, WAP-1,WAP-2, GCP-1, GCP-2, α-chemokine receptors: CXCR1, CXCR2, CXCR3, CXCR4,CXCR5, CXCR6, CXCR7 β-chemokine receptors: CCR1, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7.

Chemotherapeutics. The FcRn binding partners may be conjugated tochemotherapy or anti-tumor agents which are effective against varioustypes of human cancers, including leukemia, lymphomas, carcinomas,sarcomas, myelomas etc., such as, doxorubicin, mitomycin, cisplatin,daunorubicin, bleomycin, actinomycin D, neocarzinostatin.

Antibodies. The FcRn binding partners of the present invention may beconjugated to antibodies including, but not limited to: (a) anti-clusterof differentiation antigen CD-1 through CD-166 and the ligands orcounter receptors for these molecules; (b) anti-cytokine antibodies,e.g., anti-IL-1 through anti-IL-18 and the receptors for thesemolecules; (c) anti-immune receptor antibodies, antibodies against Tcell receptors, major histocompatibility complexes I and II, B cellreceptors, selectin killer inhibitory receptors, killer activatingreceptors, OX-40, MadCAM-1, Gly-CAM1, integrins, cadherens,sialoadherens, Fas, CTLA-4, Fcγ-receptors, Fcα-receptors, Fcε-receptors,Fcμ-receptors, and their ligands; (d) anti-metalloproteinase antibodies,e.g., collagenase, MMP-1 through MMP-8, TIMP-1, TIMP-2; anti-celllysis/proinflammatory molecules, e.g., perforin, complement components,prostanoids, nitron oxide, thromboxanes; and (e) anti-adhesionmolecules, e.g., carcioembryonic antigens, lamins, fibronectins.

Antiviral Agents. The FcRn binding partners may be conjugated toantiviral agents such as reverse transcriptase inhibitors and nucleosideanalogs, e.g. ddI, ddC, 3TC, ddA, AZT; protease inhibitors, e.g.,Invirase, ABT-538; inhibitors of in RNA processing, e.g., ribavirin.

Specific examples of known therapeutics which may be delivered via aFcRn binding partner include, but are not limited to:

(a) Capoten, Monopril, Pravachol, Avapro, Plavix, Cefzil,Duricef/Ultracef, Azactam, Videx, Zerit, Maxipime, VePesid, Paraplatin,Platinol, Taxol, UFT, Buspar, Serzone, Stadol NS, Estrace, Glucophage(Bristol-Myers Squibb);

(b) Ceclor, Lorabid, Dynabac, Prozac, Darvon, Permax, Zyprexa, Humalog,Axid, Gemzar, Evista (Eli Lily);

(c) Vasotec/Vaseretic, Mevacor, Zocor, Prinivil/Prinizide, Plendil,Cozaar/Hyzaar, Pepcid, Prilosec, Primaxin, Noroxin, Recombivax HB,Varivax, Timoptic/XE, Trusopt, Proscar, Fosamax, Sinemet, Crixivan,Propecia, Vioxx, Singulair, Maxalt, Ivermectin (Merck & Co.);

(d) Diflucan, Unasyn, Sulperazon, Zithromax, Trovan, Procardia XL,Cardura, Norvasc, Dofetilide, Feldene, Zoloft, Zeldox, Glucotrol XL,Zyrtec, Eletriptan, Viagra, Droloxifene, Aricept, Lipitor (Pfizer);

(e) Vantin, Rescriptor, Vistide, Genotropin, Micronase/Glyn./Glyb.,Fragmin, Total Medrol, Xanax/alprazolam, Sermion, Halcion/triazolam,Freedox, Dostinex, Edronax, Mirapex, Pharmorubicin, Adriamycin,Camptosar, Remisar, Depo-Provera, Caverject, Detrusitol, Estring,Healon, Xalatan, Rogaine (Pharmacia & Upjohn);

(f) Lopid, Accrupil, Dilantin, Cognex, Neurontin, Loestrin, Dilzem,Fempatch, Estrostep, Rezulin, Lipitor, Omnicef, FemHRT, Suramin,Clinafloxacin (Warner Lambert).

Further examples of therapeutic agents which may be delivered by theFcRn binding partners of the present invention may be found in: Goodmanand Gilman's The Pharmacological Basis of Therapeutics. 9th ed.McGraw-Hill 1996, incorporated herein by reference in its entirety.

5.4. Pharmaceutical Formulations for Vaccine Delivery

When administered, the conjugates of the present invention areadministered in pharmaceutically acceptable preparations. Suchpreparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines, and optionally other therapeutic agents. Thus, “cocktails”including the conjugates and the agents are contemplated. The agentsthemselves may be conjugated to FcRn binding partners to enhancedelivery of the agents across the epithelial barriers.

The conjugates of the invention may be administered in a purified formor in the form of a pharmaceutically acceptable salt. When used inmedicine the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmaceutically acceptable saltsinclude, but are not limited to, those prepared from the followingacids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic,acetic, salicylic, p-toluene sulphonic, tartaric, citric, methanesulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, andbenzene sulphonic. Also, pharmaceutically acceptable salts can beprepared as alkyline metal or alkyline earth salts, such as sodium,potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and salt (1-2% W/V);citric acid and a salt (1-3% W/V); boric acid and a salt (0.5-2:5,% WN);sodium bicarbonate (0.5-1.0% W/V); and phosphoric acid and a salt(0.8-2% W/V). Suitable preservatives include benzalkonium chloride(0.003-0.03% W/V); chlotubutanol (0.3-0.9% W/V); parabens (0.01-0.25%W/V) and thimerosal (0.004-0.02% W/V).

The term “carrier” as used herein, and described more fully below, meansone or more solid or liquid filler, dilutants or encapsulatingsubstances which are suitable for administration to a human or othermammal. The “carrier” may be an organic or inorganic ingredient, naturalor synthetic, with which the active ingredient is combined to facilitateadministration.

The components of the pharmaceutical compositions are capable of beingcommingled with the conjugates of the present invention, and with eachother, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy. The componentsof oral drug formulations include diluents, binders, lubricants,glidants, disintegrants, coloring agents and flavoring agents.Encapsulating substances for the preparation of enteric-coated oralformulations include cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate and methacrylic acidester copolymers. Solid oral formulations such as capsules or tabletsare preferred. Elixirs and syrups also are well known oral formulations.The components of aerosol formulations include solubilized activeingredients, antioxidants, solvent blends and propellants for solutionformulations, and micronized and suspended active ingredients,dispersing agents and propellants for suspension formulations. The oral,aerosol and nasal formulations of the invention can be distinguishedfrom injectable preparations of the prior art because such formulationsmay be nonaseptic, whereas injectable preparations must be aseptic. Theterm “adjuvant” is intended to include any substance which isincorporated into or administered simultaneously with the conjugates ofthe invention and which nonspecifically potentiates the immune responsein the subject. Adjuvants include aluminum compounds, e.g., gels,aluminum hydroxide and aluminum phosphate, and Freund's complete orincomplete adjuvant (in which the conjugate is incorporated in theaqueous phase of a stabilized water in paraffin oil emulsion). Theparaffin oil may be replaced with different types of oils, e.g.,squalene or peanut oil. Other materials with adjuvant properties includeBCG (attenuated Mycobacterium tuberculosis), calcium phosphate,levamisole, isoprinosine, polyanions (e.g., poly A:U) leutinan,pertussis toxin, cholera toxin, lipid A, saponins and peptides, e.g.muramyl dipeptide. Rare earth salts, e.g., lanthanum and cerium, mayalso be used as adjuvants. The amount of adjuvants depends on thesubject and the particular conjugate used and can be readily determinedby one skilled in the art without undue experimentation.

Other supplementary immune potentiating agents, such as cytokines, maybe delivered in conjunction with the conjugates of the invention. Thecytokines contemplated are those that will enhance the beneficialeffects that result from administering the immunomodulators according tothe invention. Cytokines are factors that support the growth andmaturation of cells, including lymphocytes. It is believed that theaddition of cytokines will augment cytokine activity stimulated in vivoby carrying out the methods of the invention. The preferred cytokinesare interleukin (IL)-1, IL-2, gamma-interferon and tumor necrosis factorα. Other useful cytokines are believed to be IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, erythropoietin, leukemiainhibitory factor, oncostatin-M, ciliary neurotrophic factor, growthhormone, prolactin, CD40-ligand, CD27-ligand, CD30-ligand,alpha-interferon, beta-interferon, and tumor necrosis factor-β. Othercytokines known to modulate T-cell activity in a manner likely to beuseful according to the invention are colony stimulating factors andgrowth factors including granulocyte and/or macrophage stimulatingfactors (GM-CSF, G-CSF and CSF-1) and platelet derived, epidermal,insulin-like, transforming and fibroblast growth factors. The selectionof the particular cytokines will depend upon the particular modulationof the immune system that is desired. The activity of cytokines onparticular cell types is known to those of ordinary skill in the art.

The precise amounts of the foregoing cytokines used in the inventionwill depend upon a variety of factors, including the conjugate selected,the dose and dose-timing selected, the mode of administration and thecharacteristics of the subject. The precise amounts selected can bedetermined without undue experimentation, particularly since a thresholdamount will be any amount which will enhance the desired immuneresponse. Thus, it is believed that nanogram to milligram amounts areuseful, depending upon the mode of delivery, but that nanogram tomicrogram amounts are likely to be most useful because physiologicallevels of cytokines are correspondingly low.

The preparations of the invention are administered in effective amounts.An effective amount is that amount of a conjugate that will alone, ortogether with further doses, stimulate an immune response as desired.This may involve the stimulation of a humoral antibody responseresulting in an increase in antibody titer in serum, improved mucosalimmunity, a clonal expansion of cytotoxic T lymphocytes or tolerance toan antigen, including a self antigen. It is believed that doses rangingfrom 1 nanogram/kilogram to 100 milligrams/kilogram, depending upon themode of administration, will be effective. The preferred range isbelieved to be between about 500 nanograms and 500 micrograms/kilogram,and most preferably between 1 microgram and 100 micrograms/kilogram. Theabsolute amount will depend upon a variety of factors, including theconjugate selected, the immune modulation desired, whether theadministration is in a single or multiple doses, and individual patientparameters including age, physical condition, size and weight. Fortreatment of a subject with a tumor the size, type, location andmetastases of the tumor may be factored in when determining the amountof conjugate to administer. These factors are well known to those ofordinary skill in the art and can be addressed with no more than routineexperimentation.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular conjugate selected,the particular condtion being treated and the dosage required fortherapeutic efficacy. The methods of this invention, generally speaking,involve delivering the conjugates of the invention to an epithelialsurface. Preferred modes of administration are oral, intrapulmonary,intrabinary and intranasal.

Compositions may be conveniently presented in unit dosage form and maybe prepared by any of the methods well known in the art of pharmacy. Allmethods include the step of bringing the conjugate into association witha carrier which constitutes one or more accessory, ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelybringing the conjugate into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the conjugates of the invention, increasingconvenience to the subject and the physician. Many types of releasedelivery systems are available and known to those of ordinary skill inthe art. They include polymer based systems such as polytactic andpolyglycolic acid, polyanhidrides and polycaprolactone; wax coatings,compressed tablets using conventional binders and excipients, and thelike. Bioadhesive polymer systems to enhance delivery of a material tothe intestinal epithelium are known and described in published PCTapplication WO 93/21906. Capsules for delivering agents to theintestinal epithelium also are described in published PCT application WO93/19660.

5.5. Pharmaceutical Formulations for Administration of Therapeutics

The pharmaceutical formulation of the invention contain the FcRn bindingpartner conjugate as the active ingredient in a pharmaceuticallyacceptable carrier suitable for administration and delivery in vivo. Inpreferred embodiments the pharmaceutical compositions of the presentinvention are formulated for oral, sublingual, buccal, intranasal andadministration by inhalation.

When administered, the conjugates of the present invention areadministered in pharmaceutically acceptable preparations. Suchpreparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, supplementary immune potentiating agents such as adjuvants andcytokines, and optionally other therapeutic agents. Thus, “cocktails”including the conjugates and the agents are contemplated. The agentsthemselves may be conjugated to FcRn binding partners to enhancedelivery of the agents across the epithelial barriers.

5.5.1. Dosages

The preferred amount of FcRn binding partner conjugates in allpharmaceutical preparations made in accordance with the presentinvention should be a therapeutically effective amount thereof which isalso a medically acceptable amount thereof. Actual dosage levels of FcRnbinding partner conjugates in the pharmaceutical compositions of thepresent invention may be varied so as to obtain an amount of FcRnbinding partner conjugates which is effective to achieve the desiredtherapeutic response for a particular patient, pharmaceuticalcomposition of FcRn binding partner conjugates, and mode ofadministration, without being toxic to the patient.

The selected dosage level and frequency of administration will dependupon a variety of factors including the route of administration, thetime of administration, the rate of excretion of the therapeuticagent(s) including FcRn binding partner conjugates, the duration of thetreatment, other drugs, compounds and/or materials used in combinationwith FcRn binding partner conjugates, the age, sex, weight, condition,general health and prior medical history of the patient being treatedand the like factors well known in the medical arts. For example, thedosage regimen is likely to vary with pregnant women, nursing mothersand children relative to healthy adults.

A physician having ordinary skill in the art can readily determine andprescribe the therapeutically effective amount of the pharmaceuticalcomposition required. For example, the physician could start doses ofFcRn binding partner conjugates employed in the pharmaceuticalcomposition of the present invention at levels lower than that requiredto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

5.5.2. Pharmaceutical Formulations

The pharmaceutical compositions of the present invention, including theFcRn binding partners conjugated to a therapeutic as the active agentare suitable preferably for oral, sublingual, and intranasal delivery.The pharmaceutical compositions are suitable for the delivery of theFcRn binding partner conjugates to epithelial barriers. Thepharmaceutical compositions may also be formulated to be suitable forparenteral, transdermal, intradermal and intravenous delivery.

The pharmaceutical compositions, containing biologically active FcRnbinding partner conjugates as the active agent, that are suitable fortransmucosal delivery via oral cavity delivery are in the form of asolid as lingual, buccal or sublingual tablets, troches. (lozenges),powders, time-release granules, pellets or the like may also be used, orin the form of a liquid as a liquid drop or drops, aerosol spray ormist, applied sublingually (under the tongue), on top of the tongue, orbuccally (between the cheek and gingiva). The rate of oral mucosalmembrane absorption of FcRn binding partner conjugates, is controlled bythe specific liquid or solid dosage formulation selected. Specificformulations allow the process of absorption to take place over asustained, but relatively short period of time, allowing for a gradualbuild up and constant blood level of the FcRn binding partnerconjugates.

For prolonged delivery, the active ingredient can be formulated as adepot preparation, for administration by implantation; e.g.,subcutaneous, intradermal, or intramuscular injection. Thus, forexample, the active ingredient may be formulated with suitable polymericor hydrophobic materials (e.g., as an emulsion in an acceptable oil) orion exchange resins, or as sparingly soluble derivatives; e.g., as asparingly soluble salt form of the FcRn binding partner conjugate.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle before useSuch liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated to give controlled release ofthe active compound. By way of example, but not by limitation, the FcRnbinding partners may be conjugated to the following therapeutics forepithelial barrier targeted delivery:

For buccal or sublingual administration, the compositions may take theform of tablets or lozenges formulated in conventional manner. Forrectal and vaginal routes of administration, the active ingredient maybe formulated as solutions (for retention enemas) suppositories orointments.

For administration by inhalation, the active ingredient can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular conjugate selected,the particular condtion being treated and the dosage required fortherapeutic efficacy. The methods of this invention, generally speakinginvolve delivering the conjugates of the invention to an epithelialsurface. Preferred modes of administration are oral, intrapulmonary,intrabinary and intranasal.

Compositions may be conveniently presented in unit dosage form and maybe prepared by any of the methods well known in the art of pharmacy. Allmethods include the step of bringing the conjugate into association witha carrier which constitutes one or more accessory, ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelybringing the conjugate into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the conjugates of the invention, increasingconvenience to the subject and the physician. Many types of releasedelivery systems are available and known to those of ordinary skill inthe art. They include polymer based systems such as polytactic andpolyglycolic acid, polyanhidrides and polycaprolactone; wax coatings,compressed tablets using conventional binders and excipients, and thelike. Bioadhesive polymer systems to enhance delivery of a material tothe intestinal epithelium are known and described in published PCTapplication WO 93/21906. Capsules for delivering agents to theintestinal epithelium also are described in published PCT application WO93/19660.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

6. EXAMPLES

Materials

Abbreviations

BSA, bovine serum albumin; CDNA, complementary deoxyribonucleic acid;CT-8, cholera toxin 6 subunit; DMEM, Dulbecco's modified Eagle's medium;DMSO, dimethyl sulfoxide; DOC, desoxycholate; ZCL, enhancedchemiluminescene, ELISA, enzyme linked immunosorbant assay; HBSS, Hanks'balanced salt solution without calcium or magnesium; HEPES,N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]; hGH, humangrowth hormone; IEC, intestinal epithelial cells; KI, potassium iodide;MHC, major histocompatibility complex; NAOH, sodium hydroxide; NH, CL,ammonium chloride; NHS-rhodamine, N-hydroxysuccinimidyl-rhodamine; RNA,ribonucleic acid; RT-PCR, reverse transcriptase-polymerase chainreaction; SATA, N-succinimdyl S-acetylthioacetate; SDS-PAGE, sodiumdodecyl sulfate-polyacrylamide gel electrophoresis; sulfo-LC-SPDP,sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamide] hexanoate;sulfo-NHS-biotin, sulfosuccinimidobiotin; sulfo-SMCC, sulfosuccinimidyl4-(N-maleimidoriethyl) cyclo-hexane-1-carboxylate.

Chemicals

cDNA Cycle Kits was purchased from Invitrogen (San Diego, Calif.). TAOpolymerase was purchased from Perkin-Elmer Cetus (Norwalk, Conn.).CircumVent™ Kits were purchased from New England Biolabs (Beverly,Mass.). Radionucleotides and radioactive chemicals were purchased fromDuPont/NEN (Boston, Mass.). HBSS and DMEM were purchased from GIBCO/LifeTechnologies (Gaithersburg, Md.). RPMI 1640 was purchased from Cellgro(Herndon, Va.). L-glutamine was purchased from Cellgro. ProteinA-Sepharose was purchased from Pharmacia Biotech (Piscataway, N.J.).Streptavidin-horseradish peroxidase, sulfo-LC-SPDP, sulfo-NHS-biotin,sulfo-SMCC, SATA and immobilized ficin were purchased from Pierce(Rockford, Ill.). Balb/c mice were purchased from Charles RiverLaboratories (Wilmington, Mass.). ECL kits were purchased from Amersham(Arlington Heights, Ill.). Plasmin, AvidChrom-protein A, proteinG-Sepharose, BSA, cholera toxin B subunit, anti-hGH antibodies and allother chemicals were purchased from Sigma (St. Louis, Mo.).

Example 1 Expression of FcRn mRNA in Human Intestinal Epithelial PrimaryCells and Cell Lines

Total RNA was extracted from adult human enterocytes by standardmethodology well known in the art (Sambrook et al., ibid.). Onemicrogram of RNA from each cell type was used as a template to preparethe cDNA substrate for reverse transcriptase-polymerase chain reaction(RT-PCR) using a CDNA Cycle Kit (invitrogen, San Diego, Calif.). Thirtycycles of PCR were performed on the cDNA using Taq polymerase(Perkin-Elmer Cetus, Norwalk, Conn.) according to the manufacturer'sinstructions using primers TGCTGGGCTGTGAACTG and CGCTTTTAGCAGTCGGAA. ThePCR cycle conditions were: denaturation at 94° C. for one minute,annealing at 55° C. for two minutes and extension at 72° C. for threeminutes. Amplification products were resolved by electrophoresis on a1.5% agarose gel and visualized by ethidium bromide staining, whichshowed the presence of the expected approximately 800 base pairamplification product in all samples except the adult colonic epithelialcells. To confirm the identity of the RT-PCR amplification product, theDNA band was excised from the agarose gel, subcloned into pCR 11(Invitrogen, San Diego, Calif.) and sequenced using a Prismdye-deoxyterminator cycle sequencing kit (Applied. Biosystems, Foster City,Calif.) using primers from both vector and human FcRn sequence. Reactionproducts were analyzed on an Applied Biosystems sequencer. The sequenceof the amplification products exactly matched the FcRn gene sequence,confirming the identity of the expressed gene.

Example 2 Detection of FcRn MRNA by Northern Blot

To confirm the expression of FcRn in human intestinal epithelial cellsand cell lines, a Northern blot was prepared using the RNA samplesprepared as described in Example 1 from adult human enterocytes, andfrom two human adenocarcinoma cell lines of colonic origin, CaCO-2 andHT-29. The RNA samples were resolved by formaldehyde/agarose gelelectrophoresis and transferred to a nylon membrane by standardprocedures (Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 1989). The membranewas probed using a ³²P-radiolabeled 120 base pair probe from the 3′untranslated region of FcRn by standard methods. Autoradiograms of theNorthern blot demonstrated the presence of the 1.5 kilobase hFcRntranscript in the enterocytes and both cell lines. Therefore, theexpression of FcRn in human adult intestinal epithelial cells and celllines was demonstrated by two different methods of RNA detection.

Example 3 Labeling and Immunoprecipitation of the MHC-Class I Related FcReceptor (FcRn) from Intestinal Epithelial Cells

The expression of FcRn in human intestinal epithelial cells wasconfirmed by immunoprecipitation of the protein. Caco-2 cells werelabeled metabolically using IIS-methionine (DuPont/NEN, Boston, Mass.)and proteins were extracted by methods well known in the art (Harlow andLane, Antibodies: A Laboratory Manual). A polyclonal rabbit anti-rat MHCclass I related FcR heavy chain specific antiserum bound toprotein-A-sephorose was used to immunoprecipitate FcRn from the cellextracts using standard methods (FcRn can be purified by wellestablished methods, Simister and Rees 1985, European J. Immunology,15:733-8, and used to Immunize rats followed by collection of serum,Harlow and Lane, supra.). Immunoprecipitates were resolved by SDS-PAGEand visualized by autoradiography. A 48 kilodalton FcRn protein wasobserved, confirming expression observed at the RNA level.

Example 4 Expression of FcRn Protein on the Cell Surface of HumanIntestinal Epithelial Cells

About 3×10⁷ HT-29 intestinal epithelial cells were detached from tissueculture plates by nonenzymatic methods and were washed four times withice cold Hanks' balanced salt solution containing no calcium ormagnesium (HBSS-, GIBCO/Life Technologies, Gaithersburg, Md.). To labelcell surface proteins, the washed cells were incubated twice for 20minutes with 1.5 mi of 0.5 mg/mi sulfo-NHS-biotin (Pierce, Rockford,Ill.) in DMSO. Labeled cells were washed five times with 50 mM NH₄C,incubated 20 minutes with 1 0 ml of RPMI 1640 (Cellgro, City, State)containing 1 mM L-glutamine (Mediatech, Washington, D.C.), and washedfour times with HBSS-. The cells were lysed, then precleared overnightwith protein A-Sepharose beads (Pharmacia Biotech, Piscataway, N.J.)using standard techniques well known in the art. SDS and desoxycholicacid (DOC) were added to the supernatants to final concentrations of0.1% and 0.5%, respectively. Lysates were precleared with normal rabbitserum and immunoprecipitated with polyclonal rabbit anti-rat MHC class Irelated FcR antibody by methods well known in the art.Immunoprecipitates were resolved by SDS-PAGE and transferred tonitrocellulose membranes. The nitrocellulose membrane was processed forincubation with 1:10,000 diluted streptavidinhorseradish peroxidase(Pierce, Rockford Ill.) as recommended by the manufacturer. The membranewas then processed for detection of bound horseradish peroxidase usingan ECL kit (Amersham, Arlington Heights, Ill.). Light emitted bycleavage of the chemiluminescent substrate was detected by exposure ofthe membrane to light-sensitive film. The film exposures showed thatFcRn was expressed on the surface of HT-29 intestinal epithelial cells.

Example 5 Functional Activity of Human FcRn on the Cell Surface ofIntestinal Epithelial Cells

To show that the FcRn expressed on the cell surface of intestinalepithelial cells was functional, Caco-2 cells and human adult jejunalintestinal epithelial cells (IECs) were tested for the ability to bindFc fragment of antibody. Caco-2 and jejunal I ECs were distributed tomicrocentrifuge tubes (2×10⁶ cells per tube) and pelleted at 2000 rpmfor 2-3 minutes at 4° C. Cell pellets were washed once in DMEMcontaining 20 mM HEPES, pH 6.0 or pH 8.0 at 4° C. and resuspended in 0.2ml of the same medium. The cell suspensions were transferred to 12 wellplates for assay. ¹²⁵1-Fc fragment (200 ng/ml, 4×10⁻⁹ M) in DMEMcontaining 20 mM HEPES, 1.0 mM Ki and 0.1% fish gelatin, pH 6.0 or pH8.0 with or without 0.5 mg/ml unlabeled human IgG (3.3×1 O−6 M) wasadded to each well. The cells were allowed to bind IgG or Fc at 37° C.for two hours in a 5% CO₂ humidified atmosphere. Cells were transferredto microcentrifuge tubes and pelleted at 2000 rpm for 2-3 minutes at 4°C. Unbound ¹²⁵I-Fc was removed by washing the cell pellets once withcold DMEM containing 20 mM HEPES, pH 6.0 or pH 8.0 at 4° C. Cells weredisrupted in 0.5 ml 0.1 M NAOH and the resulting solution transferred toscintillation vials. ¹²⁵I was quantified using a CliniGamma 1272 gammacounter (LKB Wallac, Piscataway, N.J.). Both Caco-2 cells and humanadult jejunum IECs specifically bound ¹²⁵I-Fc at pH 6.0 but not at pH8.0, demonstrating functional pH-dependent binding as observed for ratneonatal FcRn and cloned human FcRn (Story et al., J. Exp. Med. 180:2377-2381; December 1994).

Example 6 Preparation of Human Immunoglobulin G

Non-specific purified immunoglobulin G from human, mouse, rat, goat,pig, cow, and other species may be purchased from commercial vendorssuch as Sigma Chemical Co., Pierce Chemical, HyClone Laboratories, ICNBiomedicals and Organon Teknika-Cappei.

Immunoglobulin G also may be isolated by ammonium sulfate precipitationof precipitation of blood serum. The protein precipitate is furtherfractionated by ion exchange chromatography or gel filtrationchromatography, to isolate substantially purified non-specific IgG. Bynon-specific IgG it is meant chat no single specificity within theantibody population or pool is dominant.

Immunoglobulin G also may be purified from blood serum by adsorption toprotein A attached to a solid support such as protein A-Sepharose(Pharmacia), AvidChrom-Protein A (Sigma), or protein G-Sepharose(Sigma). Other methods of purification of IgG are well known to personsskilled in the art and may be used for the purpose of isolation ofnon-specific IgG.

Example 7 Preparation of Human lmmuglobulin G

To prepare the Fc fragments of human IgG, IgG isolated as in example 6is subjected to digestion with immobilized papain (Pierce) according tothe manufacturer's recommended protocol. Other proteases that digest IgGto produce intact Fc fragments that can bind to Fc receptors, e.g.plasmin (Sigma) or immobilized ficin (Pierce), are known to skilledartisans and may be used to prepare Fc fragments. The digestedimmunoglobulin then is incubated with an affinity matrix such as proteinA-Sepharose or protein G-Sepharose. Non-binding portions of IgG areeluted from the affinity matrix by extensive washing in batch or columnformat. Fc fragments of IgG then are eluted by addition of a buffer thatis incompatible with Fc-adsorbent binding. Other methodologies effectivein the purification of Fc fragments also may be employed.

Example 8 Conjugation of Compounds to Human Immunoglobulin Fc-Fragments

To deliver compounds via the FcRn transport mechanism, such compoundscan be coupled to whole IgG or Fc fragments. The chemistry ofcross-linking and effective reagents for such purposes are well known inthe art. The nature of the crosslinking reagent used to conjugate wholeIgG or Fc fragments and the compound to be delivered is not restrictedby the invention. Any crosslinking agent may be used provided that a)the activity of the compound is retained, and b) binding by the FcRn ofthe Fc portion of the conjugate is not adversely affected.

An example of an effective one-step crosslinking of Fc and a compound isoxidation of Fc with sodium periodate in sodium phosphate buffer for 30minutes at room temperature, followed by overnight incubation at 4° C.with the compound to be conjugated. Conjugation also may be performed byderivatizing both the compound and Fc fragments with suffosuccinimidyl6-[3-(2-pyridyldithio)propionamidel hexanoate (sulfo-LC-SPDP, Pierce)for 18 hours at room temperature. Conjugates also may be prepared byderivatizing Fc fragments and the desired compound to be delivered withdifferent crosslinking reagents that will subsequently form a covalentlinkage. An example of this reaction is derivatization of Fc fragmentswith sulfosuccinimidyl 4-(N-maleimidomethyl) cyclo-hexane-1-carboxylate(Sulfo-SMCC, Pierce) and the compound to be conjugated to Fc isthiolated with N-succinimidyl S-acetylthioacetate (SATA). Thederivatized components are purified free of crosslinker and combined atroom temperature for one hour to allow crosslinking. Other crosslinkingreagents comprising aldehyde, imide, cyano, halogen, carboxyl, activatedcarboxyl, anhydride and maleimide functional groups are known to personsof ordinary skill in the art and also may be used for conjugation ofcompounds to Fc fragments. The choice of cross-linking reagent will, ofcourse, depend on the nature of the compound desired to be conjugated toFc. The crosslinking reagents described above are effective forprotein-protein conjugations. If the compound to be conjugated is acarbohydrate or has a carbohydrate moiety, then heterobifunctionalcrosslinking reagents such as ABH, M2C2H, MPBH and PDPH are useful forconjugation with a proteinaceous FcRn binding molecule (Pierce ChemicalCo., Rockford, Ill.). Another method of conjugating proteins andcarbohydrates is disclosed by Brumeanu et al. (Genetic Engineering News,Oct. 1, 1995, p. 16). If the compound to be conjugated is a lipid or hasa lipid moiety which is convenient as a site of conjugation for the FcRnbinding molecule, then crosslinkers such as SPDP, SMPB and derivativesthereof may be used (Pierce Chemical Co., Rockford, Ill.). It is alsopossible to conjugate any molecule which is to be delivered bynoncovalent means. One convenient way for achieving noncovalentconjugation is to raise antibodies to the compound to be delivered, suchas monoclonal antibodies, by methods well known in the art, and select amonoclonal antibody having the correct Fc region and desired antigenbinding properties. The antigen to be delivered is then prebound to themonoclonal antibody carrier. In all of the above crosslinking reactionsit is important to purify the derivatized compounds free of crosslinkingreagent. It is important also to purify the final conjugatesubstantially free of unconjugated reactants. Purification may beachieved by affinity, gel filtration or ion exchange chromatographybased on the properties of either component of the conjugate. Aparticularly preferred method is an initial affinity purification stepusing protein A-Sepharose to retain Fc and Fc-compound conjugates,followed by gel filtration or ion exchange chromatography based on themass, size or charge of the Fc conjugate. The initial step of thispurification scheme ensures that the conjugate will bind to FcRn whichis an essential requirement of the invention.

Example 9 IgG-Facilitated Delivery of Foreign Antigen Across theIntestinal Epithelial Barrier

To test the ability of Fc binding partner-antigen conjugates to betransported across epithelial barriers, foreign antigens are conjugatedto IgG molecules for administration to mice. A convenient foreignantigen is the fluorescent dye rhodamine, since it may be visualized infrozen semi-thin sections of intestinal epithelium. Rhodamine iscovalently linked to non-specific mouse IgG, prepared as described inExample 6, cholera toxin B subunit (Sigma) and ovalbumin (Sigma) byincubation with succinyl-rhodamine (Molecular Probes, Eugene, Oreg.) asrecommended by the manufacturer. The IgG-rhodamine conjugate is purifiedby protein G-Sepharose affinity chromatography. After dialysis to removeunconjugated succinyl-rhodamine, cholera toxin B (CT-B)-rhodamine andovalbumin-rhodamine conjugates are purified by gel filtrations or ionexchange chromatography. Fractions of the eluate are assayed for thepresence of conjugates by determining fluorescence. Functional bindingof the IgG and CT-B subunit conjugates may be tested by binding to FcRnand ganglioside GM1, respectively. Cholera toxin B-rhodamine andovalbuminrhodamine serve as positive and negative controls,respectively.

Balb/c mice are administered 0.2 nanomoles of the three rhodamineconjugates described above, with or without 0.2 nanomoles unlabeledcholera toxin as a non-specific adjuvant, by intragastric administrationin the presence of 75 micromoles NaHCO₃ and 20 mg/mi soybean trypsininhibitor to inhibit gastric degradation. After 6 hours the mice aresacrificed and intestine is removed, frozen and processed for semi-thinsectioning. Sections of the intestinal epithelium are illuminated on afluorescent microscope and examined for intracellular fluorescence. Thepresence of fluorescence in intestinal epithelial cells of mice fedIgG-rhodamine indicates that the IgG conjugates are effectivelytransported in an apical to basolateral direction across the intestinalepithelial barrier. FcRn is capable of transporting immunogens asconjugates with FcRn binding partners.

Example 10 Mouse Mucosal Immune Response to Orally Delivered Antigen-IgGConjugate Via FcRn-Mediated Transcytosis

Transgenic mice homozygous for deletion of β2-microglobulin (a criticalcomponent of Fc-receptor function) and their normal wild-type littermates are used for studies of generation of a mucosal immune response.If rhodamine-IgG elicits a mucosal immune response by binding to apicalmembrane Fc receptors, a positive immune response should be found inwild-type but not β2-microglobulin “knockout” mice. In contrast,rhodamine-cholera toxin B subunit (CT-6) should elicit a positive immuneresponse in both wild type and—knockout” mice as transcytosis of CT-6across the epithelial barrier is not dependent on binding to apicalmembrane Fc receptors. Rhodamine-ovalbumin does,not enter transcytoticvesicles (but may enter intestinal epithelia by fluid phase endocytosis)and should not elicit an immune response in any mice.

Three groups of wild type and β₂-microglobulin knockout mice are orallyimmunized with the three rhodamine conjugates described in Example 9.Parallel experiments are conducted with the addition of 0.2 nanomoles ofcholera toxin as non-specific adjuvant. Equimolar quantities of therhodamine conjugates are administered intragastrically. The mice are“immunized” by this method every ten days for a total of three times.Two weeks after the third oral immunization the mice are sacrificed andthe rhodamine-specific immune response is determined by ELISA on gutsecretions and serum by standard methodology. Anti-rhodamine serumimmunoglobulins are most evident in the wild type mice fed rhodamineconjugates of CT-B and IgG. Knockout mice lacking 112-microglobulingenerate a mucosal immune response to rhodamine-CT-B but not torhodamine-IgG, indicating that receptor-mediated transcytosis plays anessential role in the mucosal immune response. The controlrhodamine-ovalbumin conjugate elicits little or no immune response ineither the wild type or the β₂-Microglobulin knockout mice.

Those skilled in the art will be able to recognize or ascertain with nomore than routine experimentation numerous equivalents to the specificproducts and processes described above. Such equivalents are consideredto be within the scope of the invention and are intended to be coveredby the following claims in which we claim:

Example 11 IgG-Facilitated Delivery of the Bioactive Substance Insulin,Across the Intestinal Epithelial Barrier

To test the ability of Fc binding partner-bioactive substance conjugatesto be transported across epithelial barriers, the bioactive substance,insulin is conjugated to IgG molecules for administration to mice. Aconvenient therapeutic agent to be delivered is insulin, as its presencein systemic circulation can be determined by measuring a decrease inblood glucose levels. The results of such an assay demonstrate theefficacy of the FcRn binding partner delivery system. Insulin iscovalently linked to non-specific mouse IgG, prepared as described inExample 6, cholera toxin B subunit (Sigma) and ovalbumin (Sigma) byincubation with succinyl-insulin (Molecular Probes, Eugene, Oreg.) asrecommended by the manufacturer. The IgG-insulin conjugate is purifiedby protein G-Sepharose affinity chromatography. After dialysis to removeunconjugated succinyl-Insulin, cholera toxin B (CT-B)-Insulin andovalbumin-Insulin conjugates are purified by gel filtrations or ionexchange chromatography. Fractions of the eluate are assayed for thepresence of conjugates by determining fluorescence. Functional bindingof the IgG and CT-B subunit conjugates may be tested by binding to FcRnand ganglioside GM1, respectively. Cholera toxin B-Insulin and ovalbuminInsulin serve as positive and negative controls, respectively.

Balb/c mice are administered 0.2 nanomoles of the three Insulincojugates described above, with or without 0.2 nanomoles unlabeledcholera toxin as a non-specific control, by oral administration in thepresence of 75 micromoles NaHCO₃ and 20 mg/mi soybean trypsin inhibitorto inhibit gastric degradation.

In Vivo Insulin Delivery

Balb/C mice of female sex were fasted for 12 hours before experiment.Each mouse was gavaged 200 μL of each preparation as described in Table9.3. Food was restored immediately after administration. Blood samplesfor glucose determination was drawn from mouse tail vein undermethoxyflurane anesthesia. Samples were drawn right before theadministration (0 hour), as well as at 1, 2, 3 and 3.5 hours postadministration of each preparation. Blood glucose level was measuredusing a One Touch® Profile Diabetes Tracking System (Lifescan, Milpitas,Calif.) with one Touch® Test strips, by applying blood to form a rounddrop which completely covered the test spot on the test strip. Readings(in mg/dL) were obtained from the meter indicating the blood glucoselevel detected.

The invention is not to be limited in scope by the specific embodimentsdescribed which are intended as single illustrations of individualaspects of the invention, and functionally equivalent methods andcomponents are within the scope of the invention. Indeed variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

All references cited herein are incorporated herein by reference for allpurposes.

1. A method for systemic delivery of an interferon across an epithelialbarrier of a mammal, comprising administering to the epithelial barrier,wherein the epithelial baffler comprises an epithelial tissue Thatexpresses neonatal Fc receptor (FcRn), an effective amount for deliveryof the interferon to systemic circulation of the mammal, of a conjugateof an FcRn binding partner and the interferon.
 2. The method of claim 1,wherein the interferon is interferon α.
 3. The method of claim 1,wherein the interferon is interferon β.
 4. The method of claim 1,wherein the interferon is interferon γ.
 5. The method of claim 1,wherein the FcRn binding partner comprises at least a fragment of IgGcomprising a complete binding region for FcRn.
 6. The method of claim 1,wherein the FcRn binding partner comprises at least a fragment of IgGspanning amino acid residue 248 of C_(H)2 through amino acid residue 436of C_(H)3.
 7. The method of claim 1, wherein the FcRn binding partner isan Fc fragment of IgG.
 8. The method of claim 1, wherein the interferonis covalently bound to the FcRn binding partner.
 9. The method of claim1, wherein the conjugate is a fusion protein.
 10. The method of claim 1,wherein the epithelial barrier is intestinal epithelial tissue.
 11. Themethod of claim 1, wherein the epithelial baiter is lung epithelialtissue.
 12. The method of claim 1, wherein the epithelial barrier is anasal mucosal surface.
 13. The method of claim 1, wherein theadministering to the epithelial barrier is administering orally to anintestinal epithelium.
 14. The method of claim 1, wherein theadministering to the epithelial barrier is administering in an aerosolto lungs.
 15. The method of claim 1, wherein the administering to theepithelial barrier is administering intranasally.
 16. The method ofclaim 1, wherein the mammal is an adult mammal.
 17. The method of claim1, wherein the mammal is a human.